FO-6979 substances and process for producing the same

ABSTRACT

(Beauveria) sp. FO-6979 (FERM BP-6681), which belongs to the genus Beauveria and is capable of producing FO-6979-M0, -M1, -M2, -M3 and -M4 substances, is cultured in a medium to thereby accumulate the FO-6979-M0, -M1, -M2, -M3 and -M4 substances in the liquid culture medium. Then the FO-6979-M0, -M1, -M2, -M3 and -M4 substances are collected from the culture medium. The substances thus obtained are less toxic, specifically inhibit acyl-Coenzyme A: cholesterol acyltransferase, and inhibit the formation of oil droplets in macrophages. Owing to these characteristics, the above substances are useful in preventing and treating human diseases caused by cholesterol accumulation.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to FO-6979 substances havinginhibitory action against acyl-CoA: cholesterol acyltransferase and aprocess for production thereof.

[0003] In the present invention, FO-6979 substances include FO-6979-M0substance, FO-6979-M1 substance, FO-6979 M2 substance, FO-6979-M3substance and FO-6979-M4 substance.

[0004] 2. Description of the Related Art

[0005] Recently, diseases caused by accumulation of cholesterol such ashyperlipidemia and arteriosclerosis of adults accompanied by improvementof food eating habit are brought into question as the modern disease.Cholesterol is converted to cholesterol ester by acyl group transfer ofcoenzyme A (hereinafter abbreviated as CoA) and accumulated in cells andblood lipoprotein. The enzyme which catalyzes such acyl group transferreaction is acyl-CoA: cholesterol O-acyltransferase (hereinafterdesignates as acyl-CoA cholesterol acyltransferase), and is deeplyinvolved in absorption of cholesterol from the intestinal tract,formation of lipoprotein in the liver and formation of foam cells in thecoronary artery (Sliskovic, D. R. and White, A. D. Trend. Pharm. Sci.12, 194-199, 1991).

[0006] Consequently, substance inhibiting acyl-CoA cholesterolacyltransferase may be useful for treatment of such diseases.

SUMMARY OF THE INVENTION

[0007] Development of drugs based on such mechanism of action isstrongly desired, and under such circumstances, providing substancehaving inhibitory action against acyl-CoA cholesterol acyltransferase isto provide new preventive and therapeutic methods for adult diseasessuch as hyperlipidemia and arteriosclerosis caused thereby and isuseful.

[0008] We have continued studies on metabolites produced bymicroorganisms, and as a result, we have found to produce substancehaving inhibitory action against acyl-CoA cholesterol acyltransferase inthe cultured medium of the microorganism strain FO-6979 isolated fromsoil. We had designated the substance as FO-6979 substance and had filedthe application of the invention of the substance under the title asnovel FO-6979 substance and a process for production thereof(JP-A-11-279195).

[0009] We have further continued studies on metabolites of the strainFO-6979 and newly found that substances having inhibitory action againstacyl-CoA cholesterol acyltransferase in the cultured medium of themicroorganism strain FO-6979. Subsequently, the substances havinginhibitory action against acyl-CoA cholesterol acyltransferase wereisolated from the cultured mass and purified, as a result, since thesubstances having such chemical structures were not known previously,the substances were designated as FO-6979-M0 substance, FO-6979-M1substance, FO-6979-M2 substance, FO-6979-M3 substance and FO-6979-M4substance (hereinafter sometimes designates as FO-6979 substances orFO-6979-M0 substance, -M1 substance, -M2 substance, -M3 substance and-M4 substance).

[0010] The present invention has completed according to such knowledge,consequently, the present substance has inhibitory action againstacyl-CoA cholesterol acyltransferase and inhibits lipid dropletformation in the macrophages, and as a result, it is highly expected toprevent adult diseases such as hyperlipidemia and arteriosclerosis basedon it.

[0011] The present invention provides novel FO-6979-M0 substance, whichis a compound represented by the following formula [I]

[0012] The present invention further provides a process for productionof novel FO-6979-M0 substance comprising culturing a microorganismbelonging to genus Beauveria and having ability to produce FO-6979-M0substance in a medium, accumulating FO-6979-M0 substance in the culturedliquid medium and isolating FO-6979-M0 substance from said culturedmass.

[0013] Beauveria sp. FO-6979 FERM BP-6681 can be used as themicroorganism having ability to produce FO-6979-M0 substance of thepresent invention, and the obtained substance has inhibitory actionsagainst acly-CoA cholesterol acyltransferase, formation ofcholesterylesters and lipid droplet formation in macrophages.

[0014] The present invention further provides novel FO-6979-M1substance, which is a compound represented by the following formula[II].

[0015] The present invention further provides a process for productionof novel FO-6979-M1 substance comprising culturing a microorganismbelonging to genus Beauveria and having ability to produce FO-6979-M1substance in a medium, accumulating FO-6979-M1 substance in the culturedliquid medium and isolating FO-6979-M1 substance from said culturedmass.

[0016] Beauveria sp. FO-6979 FERM BP-6681 can be used as themicroorganism having ability to produce FO-6979-M1 substance of thepresent invention, and the obtained substance has inhibitory actionsagainst acyl-CoA cholesterol acyltransferase, formation ofcholesterylesters and lipid droplet formation in macrophages.

[0017] The present invention further provides novel FO-6979-M2substance, which is a compound represented by the following formula[III]

[0018] The present invention further provides a process for productionof novel FO-6979-M2 substance comprising culturing a microorganismbelonging to genus Beauveria and having ability to produce FO-6979-M2substance in a medium, accumulating FO-6979-M2 substance in the culturedliquid medium and isolating FO-6979-M2 substance from said culturedmass.

[0019] Beauveria sp. FO-6979 FERM BP-6681 can be used as themicroorganism having ability to produce FO-6979-M2 substance of thepresent invention, and the obtained substance has inhibitory actionsagainst acyl-CoA cholesterol acyltransferase, formation ofcholesterylesters and lipid droplet formation in macrophages.

[0020] The present invention further provides novel FO-6979-M3substance, which is a compound represented by the following formula [IV]

[0021] The present invention further provides a process for productionof novel FO-6979-M3 substance comprising culturing a microorganismbelonging to genus Beauveria and having ability to produce FO-6979-M3substance in a medium, accumulating FO-6979-M3 substance in the culturedliquid medium and isolating FO-6979-M3 substance from said culturedmass.

[0022] Beauveria sp. FO-6979 FERM BP-6681 can be used as themicroorganism having ability to produce FO-6979-M3 substance of thepresent invention, and the obtained substance has inhibitory actionsagainst acyl-CoA cholesterol acyltransferase, formation ofcholesterylesters and lipid droplet formation in macrophages.

[0023] The present invention further provides novel FO-6979-M4substance, which is a compound represented by the following formula [V]

[0024] The present invention further provides a process for productionof novel FO-6979-M4 substance comprising culturing a microorganismbelonging to genus Beauveria and having ability to produce FO-6979-M4substance in a medium, accumulating FO-6979-M4 substance in the culturedliquid medium and isolating FO-6979-M4 substance from said culturedmass.

[0025] Beauveria sp. FO-6979 FERM BP-6681 can be used as themicroorganism having ability to produce FO-6979-M4 substance of thepresent invention, and the obtained substance has inhibitory actionsagainst acyl-CoA cholesterol acyltransferase, formation ofcholesterylesters and lipid droplet formation in macrophages.

[0026] The microorganism having ability to produce the above-describedFO-6979-M0 substance, -M1 substance, -M2 substance, -M3 substance and-M4 substance (hereinafter designates as “FO-6979 substance producingmicroorganism”) belongs to genus Beauveria sp., and Beauveria sp.FO-6979 strain isolated from soil by the inventors of the presentinvention is an example of the strain which can be used most effectivelyin the present invention. Taxonomical properties of the strain FO-6979are as follows.

[0027] 1. Taxonomical Properties

[0028] This strain has good growth on media such as potato glucose agar,cornmeal agar, malt extract agar and Miura agar with good growth ofconidiospores. In addition, good growth on yeast extract soluble starchagar medium is observed, but no bearing of conidiospores is observed.Microscopic observation of conidiospores grown on the cornmeal agarmedium indicates transparent hyphae with septa. The conidiophores wereborn directly from vegetable hyphae or through short branches. Basalpart of conidiospore is spherical or ventricose in size, 2.0-3.3×2.5-3.7μm. The apex is narrowly grown with zigzag shape depending on formationof conidiophores to the length of 12-20 μm. Conidiophore is budded onthe small spike (approximately 1 μm.) and is spherical or wideellipsoidal, sometimes spiky base with size, 1.8-2.5×2.5-3.3 μm.

[0029] 2. Culturing Properties on Various Media

[0030] Macroscopic observations of the strain cultured at 25° C. for 14days are shown hereinbelow. On the following media, no formation ofsclerotia or sclerotium-like structure is observed. Medium Growthcondition color tone of Color tone of on medium surface of everse ofSoluble (diameter of colony) colony colony pigment Potate glucose agarGood (40 mm) White Pale yellowish None Floccose, plane, white partlyridged penumbra, smooth penumbra Cornmeal agar Good (54 mm) White Paleyellowish None Floccose, plane, white-pale tangled thread-like orangepenumbra Malt extract agar Good (32 mm) White Pale brown None Floccose,ridged center, smooth penumbra Miura agar Good (60 mm) White White Nonepowdery, plane, smooth penumbra Yeast extract soluble starch agar Good(53 mm) White Pale yellowish None velvety, ridged, white-pale smoothpenumbra orange

[0031] 3. Physiological and Ecological Properties

[0032] (1) Optimum Growth Condition:

[0033] Optimum growth condition of this strain is pH 4-7 at 15-30° C.

[0034] (2) Growth Range:

[0035] Growth range of this strain is pH 4-10 at 11-32° C.

[0036] (3) Differentiation of aerobic and unaerobic growth: aerobic

[0037] According to the above mentioned taxonomical properties,culturing properties and physiological properties of this strainFO-6979, the strain was compared with known microorganism strains. Thisstrain was identified as the strain belonging to genus Beauveria, andwas referred to Beauveria sp. FO-6979. This strain was deposited in Mar.18, 1998 in the National Institute of Bioscience and Human-Technology,Higashi 1-1-3, Tsukuba-shi, Ibaraki-ken, Japan as the depository numberFERM P-16716. This strain was later accepted the request for transferbased on the Budapest Treaty from the original depository in Mar. 16,1999 by International Patent Organism Depository, National Institute ofAdvanced Industrial Science and Technology, AIST Tsukuba Center 6, 1-1,Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan. The permanentdepository number is FERM BP-6681.

[0038] Preferable example of FO-6979 substance producing strain used inthe present invention is the strain Beauveria sp. FO-6979 herein-beforementioned. As is well known, the taxonomical properties ofmicroorganisms are generally very easily changed by mutation and are notconstant, and are easily mutated by natural or conventional artificialmutation techniques such as ultraviolet irradiation or use of mutagenicagent, e.g. N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfonate, etc. Consequently, all strains belonging to genusBeauveria and having ability to produce FO-6979-M0 substance, -M1substance, -M2 substance, -M3 substance and -M4 substance, includingsuch artificial mutant strains and natural mutant strains, can be usedin the present invention. In addition, all strains belonging to genusBeauveria and having ability to produce FO-6979-M0 substance, -M1substance, -M2 substance, -M3 substance and -M4 substance, includingstrains mutated by cell engineering technology such as cell fusion, genemanipulation, etc., can be used in the present invention.

[0039] In practice of the present invention, at first, FO-6979 substanceproducing strain belonging to genus Beauveria is cultured in a medium.Nutrient sources preferably used for production of the above-mentionedFO-6979-M0 substance, -M1 substance, -M2 substance, -M3 substance and-M4 substance are assimilable carbon sources for microorganism,digestible nitrogen sources and, if necessary inorganic salts andvitamins can be used as nutritional medium.

[0040] Examples of carbon sources are sugars such as glucose, fructose,maltose, lactose, galactose, dextrin and starch and vegetable oils suchas soybean oil, and are used independent or in combination.

[0041] Examples of nitrogen sources are peptone, yeast extract, meatextract, soybean powder, cotton seed powder, corn steep liquor, maltextract, casein, amino acids, urea, ammonium salts and nitrates, and areused independent or in combination. If necessary, salts such asphosphate, magnesium, calcium, sodium and potassium, heavy metal saltssuch as iron, manganese, copper, cobalt and zinc, vitamins and othermaterials preferable for production of FO-6979-M0 substance, -M1substance, -M2 substance, -M3 substance and -M4 substance are optionallyadded.

[0042] In the culture, anti-foaming agents such as liquid paraffin,animal oil, vegetable oil, silicone, surface active agents, etc. can beadded if necessary when foaming occurs. The culture can be performed byliquid or solid culture if containing the above nutrient sources, andgenerally the culture is preferable performed by the liquid culture. Inthe small culture, the culture using flask is preferable. In the largescale production using the large tank, in order to prevent delay ofgrowth of microorganism in the production process, the production strainis inoculated and cultured initially in relatively small amount ofculture medium, subsequently the cultured mass is transferred into thelarge tank and the cultivation is preferable continued. In this case,compositions of the medium used in the pre culture and the medium usedin the production culture can be identical or different if necessary.

[0043] In the culture under aeration stirring condition, conventionalmeans, for example, agitation using propeller and other mechanicalstirring, rotation or shaking in fermenter, treating with pumping andblowing air can be applied. Air for aeration should be sterilized.Culturing temperature can be applied within ranges in the production ofFO-6979-M0 substance, -M1 substance, -M2 substance, -M3 substance and-M4 substance by FO 6979 substance producing strain, and the cultivationis performed usually at 20-30° C., preferably at 27° C. Culturing pH isusually 5-8, preferably about pH 7. Culturing temperature depends onculturing condition and is usually for 10-20 days.

[0044] The thus obtained FO-6979-M0 substance, -M1 substance, -M2substance, -M3 substance and -M4 substance exist in the culturedmicroorganism cells and the culture filtrate. Isolation of FO-6979-M0substance, -M1 substance, -M2 substance, -M3 substance and -M4 substancefrom the cultured mass can be performed by extracting the whole culturedmass with water miscible organic solvent such as aceton, distilling offthe organic solvent from the extract in vacuo, and extracting theresidue with water immiscible organic solvent such as ethyl acetate. Inaddition to the above extraction procedure, conventional methods usedfor isolation of fat-soluble substances such as adsorptionchromatography, gel filtration chromatography, thin-layerchromatography, counter current chromatography and high performanceliquid chromatography can be used optionally in combination orrepeatedly to isolate and purify FO-6979-M0 substance, -M1 substance,-M2 substance, -M3 substance and -M4 substance. Physicochemicalproperties of FO-6979 substances of the present invention are explainedhereinbelow.

[0045] 1. FO-6979-M0 Substance

[0046] (1) Nature: white powder.

[0047] (2) Melting point: 244-246° C.

[0048] (3) Molecular formula: C₂₂H₃₉N₃O₅, FAB-MS (m/z): 426 [M+H]⁺, 448[M+Na]⁺, HRFAB-MS (m/z) MF+Na, C₂₂H₃₉N₃O₅Na, Calculated: 448.27873,Found: 448.2787.

[0049] (4) Molecular weight: 425 (fast atom bombardment massspectrometry).

[0050] (5) Ultraviolet spectrum: maximum absorption, λmax 209 nm (CH₃OH,log ε=18216).

[0051] (6) Infrared spectrum (KBr Tablet): maximum absorption, νmax1535, 1639, 1683, 1724 cm⁻¹.

[0052] (7) Specific rotation: [α]_(D) ²³=−440 (c=0.41,chloroform:methanol=2:1).

[0053] (8) Solubility in solvent: soluble in methanol, benzene,chloroform and ethyl acetate. slight soluble in water and hexane.

[0054] (9) Color reaction: positive for sulfaric acid andphosphormolybdic acid.

[0055] (10) Grouping for acidic, neutral and basic: Neutral substance.

[0056] (11) ¹H-nuclear magnetic resonance spectrum [XL-400 (Varian Inc.,Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=1:2]: As shown in FIG. 1.

[0057] (12) ¹³C-nuclear magnetic resonance spectrum [XL-400 (VarianInc., Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=2:1]: Two signals at 13.4, 14.6,15.0, 18.2, 18.4, 18.5, 18.7, 22.5, 27.7, 29.1, 30.1, 30.4, 35.1, 35.4,49.2, 60.2, 62.1, 76.3, 169.0, 171.4, 171.8 and 171.9 ppm.

[0058] As described hereinabove, as the results of examining variousphysicochemical properties and spectral data of the present FO-6979-M0substance, FO-6979-M0 substance was determined as having chemicalstructure represented by the formula [I]

[0059] 2. FO-6979-M1 Substance

[0060] (1) Nature: white powder.

[0061] (2) Melting point: 240-242° C.

[0062] (3) Molecular formula: C₂₃H₄₁N₃O₅, FAB-MS (m/z): 440 [M+H]⁺, 462[M+Na]⁺; HRFAB-MS (m/z) MF+Na, C₂₃H₄₁N₃O₅Na, Calculated: 462.29438,Found: 462.2944.

[0063] (4) Molecular weight: 439 (fast atom bombardment massspectrometry).

[0064] (5) Ultraviolet spectrum: maximum absorption, λmax 208 nm (CH₃OH,log ε=24100).

[0065] (6) Infrared spectrum (KBr Tablet): maximum absorption, νmax1537, 1639, 1681, 1724 cm⁻¹.

[0066] (7) Specific rotation: [α]_(D) ²³=−37° (c=0.5,chloroform:methanol=2:1).

[0067] (8) Solubility in solvent: soluble in methanol, benzene,chloroform and ethyl acetate. slightly soluble in water and hexane.

[0068] (9) Color reaction: positive for sulfuric acid andphosphormolybdic acid.

[0069] (10) Grouping for acidic, neutral and basic: Neutral substance.

[0070] (11) ¹H-nuclear magnetic resonance spectrum [XL-400 (Varian Inc.,Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=1:2]: As shown in FIG. 2.

[0071] (12) ¹³C-nuclear magnetic resonance spectrum [XL-400 (VarianInc., Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=1:2]: Twenty-three signals at 10.5,13.6, 14.3, 14.7, 15.1, 18.7, 18.9, 23.0, 25.9, 28.2, 29.6, 29.8, 30.5,35.7, 35.8, 37.0, 49.6, 59.5, 76.8, 169.6, 172.1, 172.3 and 172.6 ppm.

[0072] As described hereinabove, as the result of examining variousphysicochemical properties and spectral data of the present FO-6979-M1substance, FO-6979-M1 substance was determined as having chemicalstructure represented by the formula [II]

[0073] 3. FO-6979-M2 Substance

[0074] (1) Nature: white powder

[0075] (2) Melting point: 243-246° C.

[0076] (3) Molecular formula: C₂₃H₄₁N₃O₅, FAB-MS (m/z): 440 [M+H]⁺, 462[M+Na]⁺, HRFAB-MS (m/z) MF+H, C₂₃H₄₂N₃O₅, Calculated:440.31242, Found:440.3124.

[0077] (4) Molecular weight: 439 (fast atom bombardment massspectrometry).

[0078] (5) Ultraviolet spectrum: maximum absorption, λmax 209 nm (CH₃OH,log ε=15027).

[0079] (6) Infrared spectrum (KBr Tablet): maximum absorption, νmax1537, 1639, 1681, 1724 cm⁻¹.

[0080] (7) Specific rotation: [α]_(D) ²⁵=−65° (c=0.47,chloroform:methanol=2:1).

[0081] (8) Solubility in solvent: Soluble in methanol, benzene,chloroform and ethyl acetate. slightly soluble in water and hexane.

[0082] (9) Color reaction: positive for sulfuric acid andphosphormolybdic acid.

[0083] (10) Grouping for acidic, neutral and basic: Neutral substance.

[0084] (11) ¹H-nuclear magnetic resonance spectrum [XL-400 (Varian Inc.,Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=1:2]: As shown in FIG. 3.

[0085] (12) ¹³C-nuclear magnetic resonance spectrum [XL-400 (VarianInc., Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=1:2]: Two signals at 14.3, 15.6,15.9, 19.4, 19.6, 22.6, 22.6, 23.7, 25.7, 29.0, 30.3, 30.4, 31.5, 36.4,36.6, 41.9, 50.2, 53.5, 77.3, 170.7, 172.6, 173.2 and 173.2 ppm.

[0086] As described hereinabove, as the results of examining variousphysicochemical properties and spectral data of the present FO-6979-M2substance. FO-6979 M2 substance was determined as having chemicalstructure represented by the formula [III]

[0087] 4. FO-6979-M3 Substance

[0088] (1) Nature: white powder.

[0089] (2) Melting point: 249-251° C.

[0090] (3) Molecular formula: C₂₆H₃₉N₃O₅, FAB-MS (m/z): 474 [M+H]⁺, 496[M+Na]⁺; HRFAB-MS (m/z) MF+H, C₂₆H₄₀N₃O₅, Calculated: 474.29677, Found:474.2968.

[0091] (4) Molecular weight: 473 (fast atom bombardment massspectrometry).

[0092] (5) Ultraviolet spectrum: maximum absorption, λmax 209 nm (CH₃OH,log ε=28044)

[0093] (6) Infrared spectrum (KBr Tablet): maximum absorption, νmax1537, 1639, 1687, 1726 cm⁻¹.

[0094] (7) Specific rotation: [α]_(D) ²³=−9.0 (c=0.20,chloroform:methanol=2:1).

[0095] (8) Solubility in solvent: soluble in methanol, benzene,chloroform and ethyl acetate. slightly soluble in water and hexane.

[0096] (9) Color reaction: positive for sulfuric acid andphosphormolybdic acid.

[0097] (10) Grouping for acidic, neutral and basic: Neutral substance.

[0098] (11) ¹H nuclear magnetic resonance spectrum [XL-400 (Varian Inc.,Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=4:1]: As shown in FIG. 4.

[0099] (12) ¹³C-nuclear magnetic resonance spectrum [XL-600 (VarianInc., Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=4:1]: Twenty-four signals (26carbons) at 13.6, 14.6, 15.2, 18.4, 18.6, 22.6, 29.2, 30.3, 30.6, 35.3,35.4, 35.7, 49.2, 56.8, 60.3, 76.3, 126.7, 128.3 (2 carbons), 128.8 (2carbons), 136.2, 169.1, 171.3, 171.9 and 183.5 ppm.

[0100] As described hereinabove, as a result of examining variousphysicochemical properties and spectral data of the presentFO-6979-M3substance, FO-6979-M3 substance was determined as havingchemical structure represented by the formula [IV]

[0101] 5. FO-6979-M4 Substance

[0102] (1) Nature: white powder.

[0103] (2) Melting point: 244-246° C.

[0104] (3) Molecular formula: C₂₅H₄₅N₃O₅, FAB-MS (m/z): 468 [M+H]⁺, 490[M+Na]⁺, HRFAB-MS (m/z) MF+H, C₂₅H₄₆N₃O₅, Calculated: 468.34372, Found:468.3437.

[0105] (4) Molecular weight: 467 (fast atom bombardment massspectrometry).

[0106] (5) Ultraviolet spectrum: maximum absorption, λmax 207 nm (CH₃OH,log ε=21400).

[0107] (6) Infrared spectrum (KBr Tablet): maximum absorption, νmax1537, 1639, 1685, 1722 cm⁻¹.

[0108] (7) Specific rotation: [α]_(D) ²⁸=−45° (c=0.30,chloroform:methanol=2:1).

[0109] (8) Solubility in solvent: soluble in methanol, benzene,chloroform and ethyl acetate. slightly soluble in water and hezane.

[0110] (9) Color reaction: positive for sulfuric acid andphosphormolybdic acid.

[0111] (10) Grouping for acidic, neutral and basic: Neutral substance.

[0112] (11) ¹H-nuclear magnetic resonance spectrum [XL-400 (Varian Inc.,Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=1:2]: As shown in FIG. 5.

[0113] (12) ¹³C-nuclear magnetic resonance spectrum [XL-400 (VarianInc., Japan) was used for measurement. Solvent:deuteriochloroform:deuteriomethanol=1:2]: Twenty-five signals at 11.3,14.3, 15.1, 15.5, 15.9, 19.4, 19.6, 23.3, 26.7, 28.2, 28.9, 30.4, 31.6,32.6, 36.5, 36.6, 37.8, 50.4, 60.4, 62.8, 77.6, 170.3, 170.3, 173.1 and173.4 ppm.

[0114] As described hereinabove, as a results of examining variousphysicochemical properties and spectral data of the presentFO-6979-M4substance. FO-6979-M4 substance was determined as havingchemical structure represented by the formula [V]

[0115] Inhibitory activities of FO 6979-M0 substance, -M1 substance, -M2substance, -M3 substance and -M4 substance of the present invention(hereinafter totally designates as FO-6979 substances) on acyl-CoA:cholesterol acyltransferase derived from mouse are explainedhereinbelow.

[0116] Acyl-CoA: cholesterol acyltransferase activity was assayed by themodified method of Uelmen, et al. (J. Biol. Chem. 270: 26192-26201,1995). A membrane fraction derived from mouse liver microsome was usedas an enzyme origin. A mouse liver was homogenized with buffer A [50 mMTris-HCL (pH 7.8), 1 mM EDTA and 1 mM phenylmethanesulfonyl fluoride]using Potter homogenizer (Tokyo-RIKO Inc.).

[0117] The homogenate was centrifuged at 12,000×g, and the supernatantsolution was ultracentrifuged at 100,000×g to obtain the microsomefraction which was prepared for a solution with protein concentration 5mg/ml by adding the buffer A. Assay of acyl-CoA: cholesterolacyltransferase activity was performed by adding enzyme 200 μg protein,200 mM bovine serumu alubumin, [1-¹⁴C] oleoyl-CoA (final concentration170 μM, 0.09 μCi) and each FO-6979 substance into the buffer A toprepare total 100 μl solution and incubated at 37° C. for 10 minutes.

[0118] Subsequently, the reaction was terminated by adding 0.5 mlethanol and added 1.5 ml hexane therein, then stirred well. Hexane layer1 ml was dried up and was spotted on TLC plate (silica gel plate,thickness 0.5 mm, Merck Co., U.S.A.) which was developed with a solventmixture, petroleum ether/diethyl ether/acetic acid (90:10:1, v/v). Thegenerated [¹⁴C] cholesteryl olate was quantitative measured using radioscanner (Ambis Co.). Results indicating concentration which inhibits 50%of Acyl-CoA: cholesterol acyltransferase activity of FO-6979 substances(-M0 substance˜-M4 substance) (IC₅₀) are shown in the following table.Compound IC₅₀ (μM) FO-6979-M0 substance >50 FO-5979-M1 substance >50FO-6979-M2 substance 50 FO-6979-M3 substance 40 FO-6979-M4 substance 50

[0119] Inhibitory action of FO-6979 substances of the present inventionon cholesteryl ester formation in mouse peritoneal macrophage isexplained.

[0120] Cholesteryl ester formation and lipid droplets formation in mouseperitoneal macrophage were performed according to a method of Namatameet al.(J. Biochem. 125: 319-327, 1999). Macrophage isolated from mouseperitoneal fluid was suspended at 2.0×10⁶ cells/ml in Dulbecco'smodified Eagle's medium containing 6.8% lipoprotein deficient serum(6.8% LPDS-DMEM) and aliquots (0.25 ml) were dispensed into a 48-wellmicroplate (Corning Co.).

[0121] The plate was incubated in a humidified 5% CO₂ atmosphereincubator at 37° C. for 2 hours, after which each plate was washed withHank's solution to remove the unattached cells. The plate was incubatedin 6.8% LPDS-DMEM for 1 hour, and each one of FO-6979 substances (2.5 μl1 methanol solution), liposome [consisting ofphosphatidylcholine/phosphatidylserine/dicetylphosphate/cholesterol=10:10:2:15(nmol) in 10 μl of 0.3 M glucose] and [1-¹⁴C] oleic acid (5 μl, 0.05μCi, 1 nmol) were added therein, then incubated further 14 hours.

[0122] After the incubation, supernatant was removed and theintracellular neutral lipids were extracted twice with adding hexane 0.6ml and isopropanol 0.4 ml. The mixture was concentrated, spotted on TLCplate (silica gel plate, Merck Co., thickness 0.5 mm) and developed andwith a mixed solvent of hexane/diethyl ether/acetia acid(70:30:1, v/v).The separated [¹⁴C] cholesteryl olate and [¹⁴C] triacylglycerol werequantitatively assayed using radio scanner (Ambis Co.). Resultsindicated that generation of [¹⁴C] cholesteryl olate was selectivelyinhibited and IC₅₀ value is shown in the following table. Compound IC₅₀( μM) FO-6979-M0 substance >25 FO-6979-M1 substance >45 (20 % inhibitionat 45 μM) FO-6979-M2 substance >25 FO-6979-M3 substance   21 FO-6979-M3substance >25

[0123] Inhibitory action of FO-6979 substances of the present inventionon lipid droplets formation in mouse peritoneal macrophage is explained.

[0124] Lipid droplets formation in mouse peritoneal macrophage wasperformed according to the method of Namatame et al. (J. Biochem.125:319-327, 1999). Macrophage isolated from mouse peritoneal fluid wassuspended at 2.0×10⁶ cells/ml in Dulbecco's modified Eagle's mediumcontaining 6.8% lipoprotein deficient serum (6.8% LPDS-DMEM) andaliquots (0.25 ml) were dispensed into a slide chamber (Nunc Co.).

[0125] The chamber was incubated in a humidified 5% CO₂ atmosphereincubator at 37° C. for 2 hours, after which the chamber was washed withHank's solution to remove the unattached cells. The chamber wasincubated in 6.8% LPDS-DMEM for 1 hour, and FO-6979-M3 substance (2.5 μlmethanol solution) and liposome [consisting ofphosphatidylcholine/phosphatidylserine/dicetylphosphate/cholesterol=10:10:2:15(nmol) in 10 μl of 0.3 M glucose] were added therein, then incubatedfurther 14 hours.

[0126] The lipid droplets and nuclei were double stained with oilred Oand hematoxylin, and observed by optical microscope (Olympus OpticalCo.,Japan). Results indicated that lipid droplets accumulated in cytoplasmunder 20 μM of FO-6979-M3 substance were decreased to about 50% ascompared with no drug addition (control), and lipid dropletsaccumulation was decreased in about 10-20% when added under 40 μM ofFO-6979-M0 substance, -M0 substance, -M1 substance,-M2 substance and -M4substance.

[0127] Next, toxicity test of FO-6979 substances of the presentinvention was explained hereinbelow.

[0128] No toxicity of FO-6979-M0 substance, -M1 substance, -M2substance, -M3 substance or -M4 substance was observed for growth ofmouse peritoneal macrophage at the final concentration of 50 μM. Notoxicity was noted when FO-6979-M0 substance, -M1 substance, -M2substance, -M3 substance and -M4 substance was administeredintraperitoneally in mice with 100 mg/kg.

[0129] As described hereinabove, FO-6979-M0 substance, -M1 substance,-M2 substance, -M3 substance and -4 substance of the present inventionare low toxicity, showing specific inhibitory action againstacyl-CoA:cholesterol acyltransferase and showing inhibitory actionagainst lipid droplets formation in macrophage, consequently these areexpected to be useful for prevention and treatment of diseases caused byaccumulation of cholesterol in human.

BRIEF DESCRIPTION OF THE FIGURE

[0130]FIG. 1 is ¹H-proton nuclear magnetic resonance spectrum(deuteriochloroform:deuteriomethanol=1:2) of FO-6979-M0 substance.

[0131]FIG. 2 is ¹H-proton nuclear magnetic resonance spectrum(deuteriochloroform:deuteriomethanol=1:2) of FO-6979-M1 substance.

[0132]FIG. 3 is ¹H-proton nuclear magnetic resonance spectrum(deuteriochloroform:deuteriomethanol=1:2) of FO-6979-M2 substance.

[0133]FIG. 4 is ¹H-proton nuclear magnetic resonance spectrum(deuteriochloroform:deuteriomethanol=1:2) of FO-6979-M3 substance.

[0134]FIG. 5 is ¹H-proton nuclear magnetic resonance spectrum(deuteriochloroform:deuteriomethanol=1:2) of FO-6979-M4 substance.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

[0135] The following example illustrates the present invention, but isnot construed to limit the invention.

[0136] A 500 ml Erlenmeyer flask containing a medium consisting ofglucose 2.0%, polypeptone 0.5%, yeast extract 0.2%, KH₂PO₄ 0.1%, agar0.1% and MgSO₄.7H₂O 0.05% dissolved in tap water 100 ml (adjusted to pH6.0) was cotton sealed and steam sterilized. Spore suspension (10⁷spores/ml) 100 μl of Beauveria sp. FO-6979 FERM BP-6681 grown on theagar medium was aseptically inoculated thereto and shake cultured at 27°C. for 3 days to obtain seed culture liquid.

[0137] A medium 20 lit. (adjusted to pH 6.7) consisting of glucose 1.0%,tryptone 0.5%, yeast extract 0.3% and agar 0.1% dissolved in tap waterin 30 lit. jar fermenter (Mitsuwa Co., Japan) was steam sterilized andthe above seed culture liquid 200 ml was aseptically inoculated thereto.This was cultured with agitation at 27° C. for 5 days. The culturedliquid 20 lit. was centrifuged at 10,000 rpm using KOKUSAN Type S-6ultracentrifuge (Kokusan Seiko Co.,Japan) to separate supernatant andmycelia. Mycelia were treated with acetone 18 lit. and filtered. Thefiltrate was concentrated in vacuo to obtain aqueous solution andprecipitate. The precipitate was washed with 200 ml hexane to obtaininsoluble crude substance 6 g. The substance was charged on a silica gel(600 g, silica gel 60, Merck Co., U.S.A.) column and gradually elutedstepwise with each 3.6 lit. of chloroform-methanol (100:0, 100:1, 100:2,100:3 and 0:100).

[0138] Eluted chloroform-methanol fraction (100:2) containing FO-6979-M1component, -M2 component and -M4 component was collected and dried invacuo to obtain white substance 168 mg. Each substance was repeatedlypurified using preparative HPLC [Apparatus: RANIN Co. ModelSD-200,Column: Shiseido CAPCELL-PAC C18 UG (20×250 mm), Detection: UV 210 nm,Flow rate: 6 ml/min., Elution: linear gradient with 50% aqueousacetonitrile up to 50 minutes, later 75% aqueous acetonitrilefor 25minutes]. FO-6979-M1 substance 47.6 mg was eluted at the peak withretention time of 45 minutes, FO-6979-M2 substance 44.5 mg was eluted atthe peak with retention time of 49 minutes and FO-6979-M4 substance 27.2mg was eluted at the peak with retention time of 77 minutes.

[0139] A medium 20 lit. (adjusted to pH 6.7) consisting of glucose 1.0%,tryptone 0.5%, yeast extract 0.3%, malt extract 0.3% and agar 0.1%dissolved in tap water in 30 lit. jar fermenter (Mitsuwa Co., Japan) wassteam sterilized and the above seed culture liquid 200 ml wasaseptically inoculated thereto, and then the culture was initiated withagitation at 27° C. After 24 hours, aqueous L-valine solution, which wassterilized by filtration, was aseptically added to the cultured liquidup to final concentration at 0.3%. This was further cultured withagitation at 27° C. for 4 days. The thus obtained cultured liquid 20lit. was centrifuged to obtain mycelia. Mycelia were treated withacetone and filtered. The filtrate was concentrated in vacuo to obtainprecipitate. The precipitate was washed with 200 ml hexane to obtaininsoluble crude substance 1.8 g. The substance was charged on a silicagel (180 g, silica gel 60, Merck Co., U.S.A.) column and graduallyeluted stepwise with each 1.1 lit. of chloroform-methanol (100:0, 100:1,100:2, 100:3 and 0:100).

[0140] The eluted fraction containing mainly of FO-6979-M3 component[the first half of eluate from chloroform-methanol (100:2) elution] andthe eluted fraction containing mainly FO-6979-M0 component [the laterhalf of eluate from chloroform-methanol (100:2) elution] were collectedand dried in vacuo to obtain white substances, 100 mg and 357 mgrespectively. Each substance was repeatedly purified using HPLC[Apparatus: RANIN Co. Model SD-200, Column: Shiseido CAPCELL-PAC C18 UG(20×250 mm), Detection: UV 210 nm, Flow rate: 6 ml/min., Elution:lineargradient with 50% aqueous acetonitrile up to 50 minutes, later75%aqueous acetonitrile for 25 minutes]. FO-6979-M0 substance 182 mg waseluted at the peak with retention time of 27 minutes. FO-6979-M3substance 10.5 mg was eluted at the peak with retention time of 52minutes.

INDUSTRIAL APPLICABILITY

[0141] As described hereinabove, FO-6979-M0 substance, -M1 substance,-M2 substance, -M3 substance and -M4 substance of the present inventionare low toxicity, showing specific inhibitory action againstacyl-CoA:cholesterol acyltransferase, inhibitory action against cholesterylesther and inhibitory action against lipid droplets formation inmacrophage, consequently these are expected to be useful for preventionand treatment of diseases caused by accumulation of cholesterol inhuman.

What is claimed is:
 1. FO-6979-M0 substance which is a compoundrepresented by the following formula [I]


2. A process for production of novel FO-6979-M0 substance comprisingculturing a microorganism to genus Beauveria and having ability toproduce FO-6979-M0 substance in a medium, accumulating FO-6979-M0substance in the cultured liquid medium and isolating FO-6979-M0substance from said cultured mass.
 3. The process according to claim 2wherein the microorganism belonging to genus Beauveria and havingability to produce FO-6979-M0 substance is Beauveria sp. FO-6979 FERMBP-6681.
 4. A microorganism belonging to genus Beauveria and havingability to produce FO-6979-M0 substance.
 5. The microorganism accordingto claim 4 wherein the microorganism belonging to genus Beauveria andhaving ability to produce FO-6979-M0 substance is Beauveria sp. FO-6979FERM BP-6681.
 6. FO-6979-M0 substance according to claim 1 which hasinhibitory action against acyl-CoA: cholesterol acyltransferase.
 7. FO6979-M0 substance according to claim 1 which has inhibitory actionagainst formation of cholesteryl ester.
 8. FO-6979-M0 substanceaccording to claim 1 which has inhibitory action against lipid dropletformation in macrophages.
 9. A FO-6979-M1 substance which is a compoundrepresented by the following formula [II]


10. A process for production of novel FO-6979-M1 substance comprisingculturing a microorganism belonging to geneus Beauveria and havingability to produce FO-6979-M1 substance in a medium, accumulatingFO-6979-M1 substance in the cultured liquid medium and isolatingFO-6979-M1substance from said cultured mass.
 11. The process accordingto claim 10 wherein the microorganism belonging to genus Beauveria andhaving ability to produce FO-6979-M1 substance is Beauveria sp. FO-6979FERM BP-6681.
 12. A microorganism belonging to genus Beauveria andhaving ability to produce FO-6979-M1 substance.
 13. The microorganismaccording to claim 12 wherein the microorganism belonging to genusBeauveria and having ability to produce FO-6979-M1 substance isBeauveria sp. FO-6979 FERM BP-6681.
 14. FO-6979-M1 substance accordingto claim 9 which has inhibitory action against acyl-CoA: cholesterolacyltransferase.
 15. FO-6979-M1 substance according to claim 9 which hasinhibitory action against formation of cholesteryl ester.
 16. FO-6979-M1substance according to claim 9 which has inhibitory action against lipiddroplet formation in macrophages.
 17. FO-6979-M2 substance which is acompound represented by the following formula [III]


18. A process for production of novel FO-6979-M2 substance comprisingculturing a microorganism belonging to genus Beauveria and havingability to produce FO-6979-M2 substance in a medium, accumulatingFO-6979-M2 substance in the cultured liquid medium and isolatingFO-6979-M2 substance from said cultured mass.
 19. The process accordingto claim 18 wherein the microorganism belonging to genus Beauveria andhaving ability to produce FO-6979-M2 substance is Beauveria sp. FO-6979FERM BP-6681.
 20. A microorganism belonging to genus Beauveria andhaving ability to produce FO-6979-M2 substance.
 21. The microorganismaccording to claim 20 wherein the microorganism belonging to genusBeauveria and having ability to produce FO-6979-M2 substance isBeauveria sp. FO-6979 FERM BP-6681.
 22. FO-6979-M2 substance accordingto claim 17 which has inhibitory action against acyl-CoA: cholesterylacyltransferase.
 23. FO-6979-M2 substance according to claim 17 whichhas inhibitory action against formation of cholesteryl ester. 24.FO-6979-M2 substance according to claim 17 which has inhibitory actionagainst lipid droplet formation in macrophages.
 25. FO-6979-M3 substancewhich is a compound represented by the following formula [IV]


26. A process for production of novel FO-6979-M3 substance comprisingculturing a microorganism belonging to genus Beauveria and havingability to produce FO-6979-M3 substance in a medium, accumulatingFO-6979-M3 substance in the cultured liquid medium and isolatingFO-6979-M3 substance from said cultured mass.
 27. The process accordingto claim 26 wherein the microorganism belonging to genus Beauveria andhaving ability to produce FO-6979-M3 substance is Beauveria sp. FO-6979FERM BP-6681.
 28. A microorganism belonging to genus Beauveria andhaving ability to produce FO-6979 M3 substance.
 29. The microorganismaccording to claim 28 wherein the microorganism belonging to genusBeauveria and having ability to produce FO-6979-M3 substance isBeauveria sp. FO-6979 FERM BP-6681.
 30. FO-6979-M3 substance accordingto claim 25 which has inhibitory action against acyl-CoA: cholesterolacyltransferase.
 31. FO 6979 M3 substance according to claim 25 whichhas inhibitory action against formation of cholesteryl ester. 32.FO-6979-M3 substance according to claim 25 which has inhibitory actionagainst lipid droplet formation in macrophages.
 33. FO-6979-M4 substancewhich is a compound represented by the following formula [V]


34. A process for production of novel FO-6979-M4 substance comprisingculturing a microorganism belonging to genus Beauveria and havingability to produce FO-6979-M4 substance in a medium, accumulatingFO-6979-M4 substance in the cultured liquid medium and isolatingFO-6979-M4 substance from said cultured mass.
 35. The process accordingto claim 34 wherein microorganism belonging to genus Beauveria andhaving ability to produce FO-6979-M4 substance is Beauveria sp. FO-6979FERM BP-6681.
 36. A microorganism belonging to genus Beauveria andhaving ability to produce FO-6979-M4 substance.
 37. The microorganismaccording to claim 36 wherein the microorganism belonging to genusBeauveria and having ability to produce FO-6979-M4 substance isBeauveria sp. FO-6979 FERM BP-6681.
 38. FO-6979-M4 substance accordingto claim 33 which has inhibitory action against acyl-CoA: cholesyerolacyltransferase.
 39. FO-6979-M4 substance according to claim 33 whichhas inhibitory action against formation of cholesteryl ester. 40.FO-6979-M4 substance according to claim 33 which has inhibitory actionagainst lipid droplet formation in macrophages.